Fluctuation Relations to Calculate Protein Redox Potentials from Molecular Dynamics Simulations.

The tunable design of protein redox potentials promises to open a range of applications in biotechnology and catalysis. Here, we introduce a method to calculate redox potential changes by combining fluctuation relations with molecular dynamics simulations. It involves the simulation of reduced and oxidized states, followed by the instantaneous conversion between them. Energy differences introduced by the perturbations are obtained using the Kubo-Onsager approach. Using a detailed fluctuation relation coupled with Bayesian inference, these are postprocessed into estimates for the redox potentials in an efficient manner. This new method, denoted MD + CB, is tested on a de novo four-helix bundle heme protein (the m4D2 "maquette") and five designed mutants, including some mutants characterized experimentally in this work. The MD + CB approach is found to perform reliably, giving redox potential shifts with reasonably good correlation (0.85) to the experimental values for the mutants. The MD + CB approach also compares well with redox potential shift predictions using a continuum electrostatic method. The estimation method employed within the MD + CB approach is straightforwardly transferable to standard equilibrium MD simulations and holds promise for redox protein engineering and design applications.

In silico study directed towards identification of the key structural features of GyrB inhibitors targeting MTB DNA gyrase: HQSAR, CoMSIA and molecular dynamics simulations.

Mycobacterium tuberculosis DNA gyrase subunit B (GyrB) has been identified as a promising target for rational drug design against fluoroquinolone drug-resistant tuberculosis. In this study, we attempted to identify the key structural feature for highly potent GyrB inhibitors through 2D-QSAR using HQSAR, 3D-QSAR using CoMSIA and molecular dynamics (MD) simulations approaches on a series of thiazole urea core derivatives. The best HQSAR and CoMSIA models based on IC50 and MIC displayed the structural basis required for good activity against both GyrB enzyme and mycobacterial cell. MD simulations and binding free energy analysis using MM-GBSA and waterswap calculations revealed that the urea core of inhibitors has the strongest interaction with Asp79 via hydrogen bond interactions. In addition, cation-pi interaction and hydrophobic interactions of the R2 substituent with Arg82 and Arg141 help to enhance the binding affinity in the GyrB ATPase binding site. Thus, the present study provides crucial structural features and a structural concept for rational design of novel DNA gyrase inhibitors with improved biological activities against both enzyme and mycobacterial cell, and with good pharmacokinetic properties and drug safety profiles.

"Pipe Organ" Inspired Air-Coupled Ultrasonic Transducers With Broader Bandwidth.

Piezoelectric micromachined ultrasonic transducers (PMUTs) are used to receive and transmit ultrasonic signals in industrial and biomedical applications. This type of transducer can be miniaturized and integrated with electronic systems since each element is small and the power requirements are low. The bandwidth of the PMUT may be narrow in some conventional designs; however, it is possible to apply modified structures to enhance this. This paper presents a methodology for improving the bandwidth of air-coupled PMUTs without sensitivity loss by connecting a number of resonating pipes of various lengths to a cavity. A prototype piezoelectric diaphragm ultrasonic transducer is presented to prove the theory. This novel device was fabricated by additive manufacturing (3-D printing), and consists of a polyvinylidene fluoride thin film over a stereolithography designed backplate. The backplate design is inspired by a pipe organ musical instrument, where the resonant frequency (pitch) of each pipe is mainly determined by its length. The -6-dB bandwidth of the "pipe organ" air-coupled transducer is 55.7% and 58.5% in transmitting and receiving modes, respectively, which is ~5 times wider than a custom-built standard device.

L718Q mutant EGFR escapes covalent inhibition by stabilizing a non-reactive conformation of the lung cancer drug osimertinib.

Osimertinib is a third-generation inhibitor approved for the treatment of non-small cell lung cancer. It overcomes resistance to first-generation inhibitors by incorporating an acrylamide group which alkylates Cys797 of EGFR T790M. The mutation of a residue in the P-loop (L718Q) was shown to cause resistance to osimertinib, but the molecular mechanism of this process is unknown. Here, we investigated the inhibitory process for EGFR T790M (susceptible to osimertinib) and EGFR T790M/L718Q (resistant to osimertinib), by modelling the chemical step (i.e., alkylation of Cys797) using QM/MM simulations and the recognition step by MD simulations coupled with free-energy calculations. The calculations indicate that L718Q has a negligible impact on both the activation energy for Cys797 alkylation and the free-energy of binding for the formation of the non-covalent complex. The results show that Gln718 affects the conformational space of the EGFR-osimertinib complex, stabilizing a conformation of acrylamide which prevents reaction with Cys797.

Spatial Correlation of Pathology and Perfusion Changes within the Cortex and White Matter in Multiple Sclerosis.

BACKGROUND AND PURPOSE: The spatial correlation between WM and cortical GM disease in multiple sclerosis is controversial and has not been previously assessed with perfusion MR imaging. We sought to determine the nature of association between lobar WM, cortical GM, volume and perfusion. MATERIALS AND METHODS: Nineteen individuals with secondary-progressive multiple sclerosis, 19 with relapsing-remitting multiple sclerosis, and 19 age-matched healthy controls were recruited. Quantitative MR perfusion imaging was used to derive CBF, CBV, and MTT within cortical GM, WM, and T2-hyperintense lesions. A 2-step multivariate linear regression (corrected for age, disease duration, and Expanded Disability Status Scale) was used to assess correlations between perfusion and volume measures in global and lobar normal-appearing WM, cortical GM, and T2-hyperintense lesions. The Bonferroni adjustment was applied as appropriate. RESULTS: Global cortical GM and WM volume was significantly reduced for each group comparison, except cortical GM volume of those with relapsing-remitting multiple sclerosis versus controls. Global and lobar cortical GM CBF and CBV were reduced in secondary-progressive multiple sclerosis compared with other groups but not for relapsing-remitting multiple sclerosis versus controls. Global and lobar WM CBF and CBV were not significantly different across groups. The distribution of lobar cortical GM and WM volume reduction was disparate, except for the occipital lobes in patients with secondary-progressive multiple sclerosis versus those with relapsing-remitting multiple sclerosis. Moderate associations were identified between lobar cortical GM and lobar normal-appearing WM volume in controls and in the left temporal lobe in relapsing-remitting multiple sclerosis. No significant associations occurred between cortical GM and WM perfusion or volume. Strong correlations were observed between cortical-GM perfusion, normal appearing WM and lesional perfusion, with respect to each global and lobar region within HC, and RRMS and SPMS patients (R2 ≤ 0.96, P < .006 and R2 ≤ 0.738, P < .006). CONCLUSIONS: The weak correlation between lobar WM and cortical GM volume loss and perfusion reduction suggests the independent pathophysiology of WM and cortical GM disease.

Wave propagation in 0-3/3-3 connectivity composites with complex microstructure.

This work presents a study of the properties of particulate composites. The whole range of particle volume fraction (0-1) and ideal 0-3, 3-3 and intermediate 0-3/3-3 connectivities are analysed. Two different approaches to produce a realistic model of the complex microstructure of the composites are considered. The first one is based on a random location of mono-dispersed particles in the matrix; while the second incorporates a size distribution of the particles based on experimental measurements. Different particle shapes are also considered. A commercial finite element package was used to study the propagation of acoustic plane waves through the composite materials. Due to the complexity of the problem, and as a first step, a two-dimensional model was adopted. The results obtained for the velocity of sound propagation from the finite element technique are compared with those from other theoretical approaches and with experimental data. The study validates the use of this technique to model acoustic wave propagation in 0-3/3-3 connectivity composites. In addition, the finite element calculations, along with the detailed description of the microstructure of the composite, provide valuable information about the micromechanics of the sample and the influence of the microstructure on macroscopic properties.

Results of the L5178Y mouse lymphoma assay and the Balb/3t3 cell in vitro transformation assay for eight phthalate esters.

Eight phthalate esters, with alcohol chain lengths of 1-11 carbon atoms and with various degrees of branching, were tested in vitro in the L5178Y mouse lymphoma mammalian cell mutation assay and in the Balb/3T3 cell transformation assay. The tests were performed as part of a voluntary testing agreement between the Chemical Manufacturers Association's Phthalate Esters Panel and the United States Environmental Protection Agency (US EPA). The esters tested were: dimethyl phthalate (DMP), di-n-butyl phthalate (DBP), butyl benzyl phthalate (BBP), di-¿n-hexyl, n-octyl, n-decyl¿ phthalate (610P), di-isononyl phthalate (DINP), di-¿heptyl, nonyl, undecyl¿ phthalate (711P), di-isodecyl phthalate (DIDP) and di-undecyl phthalate (DUP). Both DMP and DBP were found to produce significant increases in the mutant frequency in the mouse lymphoma assay in the presence but not in the absence of an Aroclor-induced rat liver activation system (S-9). Ester 610P gave equivocal results in the mouse lymphoma assay in the presence and absence of rat liver S-9. There was no indication of mutagenic potential for any of the other test materials in the mouse lymphoma assay, and none of the test materials increased transformation frequency in the Balb/3T3 cell transformation assay. Aldehyde metabolites of the de-esterified alcohols are postulated to play a role in the positive results for DMP and DBP.

Combined quantum mechanical and molecular mechanical reaction pathway calculation for aromatic hydroxylation by p-hydroxybenzoate-3-hydroxylase.

The reaction pathway for the aromatic 3-hydroxylation of p-hydroxybenzoate by the reactive C4a-hydroperoxyflavin cofactor intermediate in p-hydroxybenzoate hydroxylase (PHBH) has been investigated by a combined quantum mechanical and molecular mechanical (QM/MM) method. A structural model for the C4a-hydroperoxyflavin intermediate in the PHBH reaction cycle was built on the basis of the crystal structure coordinates of the enzyme-substrate complex. A reaction pathway for the subsequent hydroxylation step was calculated by imposing a reaction coordinate that involves cleavage of the peroxide oxygen-oxygen bond and formation of the carbon-oxygen bond between the C3 atom of the substrate and the distal oxygen of the peroxide moiety of the cofactor. The geometric changes and the Mulliken charge distributions along the calculated reaction pathway are in line with an electrophilic aromatic substitution type of mechanism. The energy barrier of the calculated reaction is considerably lower when the substrate hydroxyl moiety is deprotonated, in comparison with the barrier found with a protonated hydroxyl moiety. This effect of the protonation state of the substrate on the calculated energy barrier supports experimental observations that deprotonation is required for hydroxylation of the substrate. A notable event in the calculated reaction pathway is a lengthening of the peroxide oxygen-oxygen bond at an intermediate stage. Further analysis of the reaction pathway indicates that this oxygen-oxygen bond elongation is accompanied by an increase in electrophilic reactivity on the distal oxygen of the peroxide moiety, which may assist the C-O bond formation in the reaction of the C4a-hydroperoxyflavin intermediate with the substrate. Analysis of the effect of individual active site residues on the reaction reveals a specific transition state stabilization by the backbone carbonyl moiety of Pro293. The crystal water 717 appears to drive the hydroxylation step through a stabilizing hydrogen bond interaction to the proximal oxygen of the C4a-hydroperoxyflavin intermediate, which increases in strength as the hydroperoxyflavin cofactor converts to the anionic (deprotonated) hydroxyflavin.

Acetyl-CoA enolization in citrate synthase: a quantum mechanical/molecular mechanical (QM/MM) study.

Citrate synthase forms citrate by deprotonation of acetyl-CoA followed by nucleophilic attack of this substrate on oxaloacetate, and subsequent hydrolysis. The rapid reaction rate is puzzling because of the instability of the postulated nucleophilic intermediate, the enolate of acetyl-CoA. As alternatives, the enol of acetyl-CoA, or an enolic intermediate sharing a proton with His-274 in a "low-barrier" hydrogen bond have been suggested. Similar problems of intermediate instability have been noted in other enzymic carbon acid deprotonation reactions. Quantum mechanical/molecular mechanical calculations of the pathway of acetyl-CoA enolization within citrate synthase support the identification of Asp-375 as the catalytic base. His-274, the proposed general acid, is found to be neutral. The acetyl-CoA enolate is more stable at the active site than the enol, and is stabilized by hydrogen bonds from His-274 and a water molecule. The conditions for formation of a low-barrier hydrogen bond do not appear to be met, and the calculated hydrogen bond stabilization in the reaction is less than the gas-phase energy, due to interactions with Asp-375 at the active site. The enolate character of the intermediate is apparently necessary for the condensation reaction to proceed efficiently.

Chemical industry voluntary test program for phthalate esters: health effects studies.

The Chemical Manufacturers' Association voluntary test program on phthalate esters is described, and the results of certain key aspects of the program are presented. Representative phthalate esters were chosen for genotoxicity testing and peroxisome proliferation screening, and di-2-ethylhexyl phthalate (DEHP) and its initial metabolic products were tested in the genotoxicity battery. A comparative metabolism study was performed with DEHP in the mouse, rat, and cynomologus monkey, together with a study of the metabolism of DEHP in the rodent at several dose levels, and after prolonged feeding. A standard test for peroxisome proliferation in the rat, employing 21 days of feeding and several end points is described, based on DEHP as a reference compound. DEHP is shown to be nongenotoxic in the test battery, and its initial major metabolites are also nongenotoxic. A nonlinear dose response with respect to the beta-oxidation of DEHP in the rodent is demonstrated. Quantitative differences exist between the mouse and rat, and the cynomologus monkey with respect to the beta-oxidation of DEHP, beta-oxidation being a much less used pathway in the monkey. The significance of these results in interpreting the hepatocellular carcinogenesis of DEHP in the Fischer 344 rat is discussed.

Characterization of a flagellar sheath protein of Vibrio cholerae.

A flagellar sheath protein of Vibrio cholerae CA401 (Inaba) was characterized. Purity of the preparation was indicated by a single band on polyacrylamide gel electrophoresis gels and on Ouchterlony plates prepared with antibody against crude sheath material. The sheath protein was composed of three polypeptides with minimal molecular weights of 61,500, 60,000, and 56,500. The presence of sheath protein on the flagellum as well as on the outer membrane of the cell was demonstrated by ferritin labeling experiments with antiserum. Sheath protein antibody reacted similarly in labeling experiments and agglutination tests with a classical Ogawa strain and two nonagglutinating V. cholerae isolates, indicating that the sheath protein may represent the common Vibrio H antigen. Antibody specific for lipopolysaccharide labeled the cell but not the sheathed flagellum, which demonstrated that the sheath is not a simple extension of the outer membrane of the cell.